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Some aspects of intermediary metabolism in the desert locust (Schistocerca gregaria Forskål)
J. Ins. Physiol., 1963, Vol. 9, pp. 463 to 468. Pergamon Press Ltd.
Printed in Great Britain
SOME ASPECTS OF INTERMEDIARY METABOLISM IN THE DESERT LOCUST (SCHISTOCERCA GREGARIA FORSIdL) W. J. W. HINES
and M. J. H. SMITH
Department of Chemical Pathology, Ring’s College Hospital Medical School, Denmark Hill, London (Received 2 March 1963)
Abstract-The incorporation and distribution of radioactivity from [“Cl glucose, [2-i%] acetate, and [l :4-“CI] succinate into the soluble metabolic intermediates of homogenates of the fat-body, head, end leg muscle of Schistocerca gregaria ForskHlhave been studied. The tissue homogenates were found to utilize the labelled substrates, but the patterns of incorporation of the radioactivity from each substrate varied with the tissue. INTRODUCTION
THE development of many insecticides during the last decade has stimulated interest in their modes of action. WINTERINGHAMand Lrrwrs (1959) have rightly emphasized that the exact manner by which an insecticide causes the death of a particular insect may be due to multiple biochemical and physiological actions rather than to some unique single lesion. It is therefore desirable to investigate as many as possible of the effects of such compounds on insect metabolism and to attempt to define them in biochemical terms. A necessary prerequisite is a detailed knowledge of insect biochemistry, but the study of this field has tended to lag behind corresponding work with mammalian tissues and micro-organisms. In recent years, the development of chromatographic methods for the separation of metabolic intermediates and the availability of radioactively labelled substrates have greatly facilitated the study of intermediary metabolism. This paper is concerned with the incorporation and subsequent distribution of radiocarbon from labelled glucose, acetate, and succinate amongst the soluble metabolic intermediates of the fat body, the nervous tissue of the head, and the leg muscle of the desert locust, Schisiocerca gregaria Forskil. MATERIALS AND METHODS The desert locust was chosen because of its large size and also because many individuals could be obtained on a single occasion from a standard population. Immature male and female insects were obtained from the Anti-Locust Research Centre, London, about 10 days after the final ecdysis and used at once. All dissections were carried out on cracked ice, and subsequent operations were performed as close to 0°C as possible. The heads were removed with scissors, the
W. J. W. Hmzs ANDM. J. H. SMITH
464
mouth-parts were cut off, and the remainder of the head was chopped up finely and homogenized with 4 volumes of cold 0.25 M sucrose in a Waring blendor. The femur muscle was removed from the hind legs as completely as possible, cut finely with scissors, and homogenized as above. The abdomen was cut open longitudinally along the mid-dorsal line, and the perivisceral fat body removed from either side of the gut with a pair of forceps and homogenized in 2 volumes of cold 0.25 M sucrose as above. Samples (50 ~1) of the homogenates were added to 75 ~1 of a solution containing 1 PC of the radioactive substrate, 5 mM MgCl,. m 6H,O, 0.15 mM adenosine triphosphate, dissolved in O-1 M phosphate buffer, pH 7.5 (GOMORI, 1955). The [‘“Cl glucose, [2-14C] acetate, and [l YI-‘~C~ succinate were obtained from the Radiochemical Centre, Amersham, Bucks. The mixtures were incubated aerobically in stoppered 5 ml tubes at 30°C with shaking for 60 min and the enzymes inactivated by the addition of 400 ~1 of boiling ethanol. After centrifuging for 5 min at 500g to remove denatured protein material, the radioactive substances in the supematants were separated by two-dimensional chromatography, visualized by radioautography, and the 14C was measured by the techniques described by SMITH and Moses (1960). RESULTS Metabolisnr of [‘“Cl glucose by locust tiwues The homogenates of all three tissues were capable of glucose (Table l), the leg muscle being the most active and as assessed by the amounts of residual radioactive glucose. corporated into trehalose by the three tissues ; they also
utilizing the labelled head the least active, Radiocarbon was informed a radioactive
TABLII I-DISTRIBUTION OF RADIOACTIVITY FROM [**Cl GLUCOSE IN SOLUBLE INTERMEDIATES OF HOMOGENATES OF LOCUSTTISSUES.&SULTs ARIIBXPRESSED AS COUNTSF%RMIN X 1o-3 Radioactive intermediate Trehalose Oligosaccharide Hexose phosphate Lactate Alanine Aspartate Glutamate Unidentified Glucose (residual substrate) Total in soluble fraction
Leg muscle
Tissue homogenate Fat-body
Head
0.1 3.5 4.8 1.0 5.6 0.1 0 0.1 5.8 15.2
1.1 1.2 2.9 0.4 3.4 4.1 0.1 0.5 9.8 13.7
1.0 3.9 0.6 0.4 3.4 0.3 1.3 0.3 13.9 11.2
oligosaccharide fraction which was not glycogen but produced on hydrolysis radioactive glucose only. The muscle and fat-body homogenates formed relatively large amounts of a hexose phosphate fraction, and ail the tissues incorporated
SOME ASPECTS OF INTERMEDIARY
METABOLISM
IN THE DESERT LOCUST
465
the isotope into lactate and alanine. The ratios alanine : lactate were approximately 6:l for the muscle and 10: 1 for the other tissues. Radioactivity was also detected in aspartate, and the fat-body and the head homogenates also formed radioactive glutamate. Metabolism of [2-‘4C]
acetate by locust tissues
The distribution of radioactivity from the labelled acetate amongst the soluble intermediates of the locust tissue homogenates is given in Table 2. The general pattern of incorporation of the isotope was similar for all three tissues except that the head homogenate formed a much greater proportion of labelled glutamate. TABLE SOLUBLE
2--DISTRIBUTION METABOLIC &SIJLTS
OF
RADIOACTIVITY
INTBRMEDIATBS
[2-“c]
FROM
OF HOMOGENATES
ARE BXPRBSSED AS COUNTS PER MIN
Radioactive intermediate Citrate Succinate Furnarate Malate Aspartate Glutamate Glutamine Alanine Lactate Proline Unidentified Total in soluble fraction
ACETATE
OF LOCUST
IN
TISSUES.
X 1o-3
Leg muscle
Tissue homogenate Fat-body
Head
O-1 0.2 0.1 0.8 0.1 4.3 o-2 l-2 l-5 0.5 0.1 9.1
O-1 0.5 0.3 1.8 0.1 2.0 0.1 0.2 1.2 0.1 0.1 6.5
0.4 1.0 0.3 1.5 0.1 9.5 0.8 1.1 3.5 0.2 0.2 18.6
Radioactivity was found in lactate and alanine, in acids of the tricarboxylic acid cycle (citrate, succinate, fumarate, and malate), and in amino acids (glutamate and aaparate), which were probably formed by transamr ‘nation reactions from their corresponding a-keto acids (a-ketoglutarate and oxaloacetate) which are also components of the tricarboxylic acid cycle. Labelled glutamine and proline were also detected in all the tissue homogenates. Metabolism of [ 1: 4-14CsJ succinate by locust times The results with the labelled succinate (Table 3) show that all three tissue homogenates were capable of utilizing this substrate by succinic dehydrogenase and metabolizing it via the tricarboxylic acid cycle. Radioactivity was found in fumarate, malate, citrate, aspartate, and glutamate. The presence of labelled glutamine confirmed the findings with the labelled acetate that glutamine synthetase activity occurred in all three tissues. The incorporation of radiocarbon into alanine and a phosphate fraction suggested that the succinate carbons had been converted to pyruvate presumably via the decarboxylation of malate or 3*
W. J. W. HINES AND M. J. H. SMITH
466 oxaloacetate.
The head homogenate again formed the largest amount of radio-
active glutamate, as in the acetate experiments, and the fat-body showed a relatively large incorporation of Cl4 into malate. TABLE %---DISTIXIBUTION OF RADIOACTIVITV FROM[t :4-“&J SUCCINATE IN IATESOF HOMOGENATES OF LOCUSTTISSUES. SOLUBLEMSTABOLICINTBRMBD &ISULTSAREEXPRESSED AS COUNTS PER MIN X lo-* Radioactive intermediate
Leg muscle
Tissue homogenate Fat-body
Head
Fumarate Malate Citrate Aspartate Glutamate Glutamine Alanine Phosphates Unidentified Succinate (residual substrate) Total in soluble fraction
1.4 4.8 0.2 0.4 2.8 0.4 2.7 0.1 0.1 20.5 33.4
3.4 16.2 0.1 0.3 2.3 0.1 2.2 0.1 0.1 25.5 50.3
0.8 4.4 0.5 0.3 5.9 0.7 2.1 0.1 0.2 15.6 30.6
DISCUSSION Studies
of isotope incorporation can provide useful data about the biochemical
pathways and the metabolic capabilities and relative importance of different tissues. Thus
the present results show that homogenates
metabolize
of locust tissues are able to
a wide range of radioactive substrates by established pathways
of
intermediary metabolism.
However, the patterns of incorporation of the radiocarbon amongst the soluble intermediates show differences between the tissues. Previous work in the locust using labelled glucose as a substrate has been almost entirely restricted to preparations of fat-body.
CLEMENTS (1959) found that
sheets of S. gregaria fat-body incorporated radiocarbon from r4C] glucose mainly into trehalose, smaller amounts being incorporated into glycogen, fat, and into an unidentified substance which was not glucose, ribose, alanine, or glutamate. CANDY and KILBY (1959) reported similar results and showed that the in Z&W biosynthesis of trehalose from glucose was increased by the addition of adenosine triphosphate
and uridine diphosphate
glucose.
They
also stated that a number
of unidentified radioactive compounds were formed from labelled glucose by locust fat-body tissue. The present results (Table 1) show that leg muscle and head homogenates are also capable of forming trehalose from glucose. In addition, radioactivity was incorporated into an oligosaccharide fraction, and all three tissue homogenates formed labelled hexose phosphates, lactate, and alanine. These findings indicate that as well as converting the glucose into the disaccharide, the locust tissues are capable of glycolytic reactions, and the pyruvate formed may be transaminated to alanine and acted on by lactate dehydrogenase to yield lactate.
SOME ASPECTS OF INTJ3RMEDIAFtY METABOLISM
IN THE DESERT LOCUST
467
The transamination reaction appeared to be quantitatively more important because the ratios of radioactive alanine to lactate varied from approximately 6:l to 10: 1, depending on the tissue. The muscle homogenate incorporated higher proportions of the radioactivity into the labelled intermediates (hexose phosphates, alanine, and lactate), associated with glycolysis, than did the other tissues. Radioactive aspartate was found in all the tissue homogenates, particularly the fat-body, and the carbon skeleton of this amino acid could be derived either via the carboxylation of pyruvate to oxaloacetate and subsequent transamination or after the entry of pyruvate carbons into the tricarboxylic acid cycle. The presence of labelled glutamate in the fat-body and head homogenates provides good evidence that glucose carbons had entered the tricarboxylic acid cycle, presumably via pyruvate and acetylcoenzyme A, to form a-ketoglutarate which was transaminated to glutamate. The formation of labelled glutamate from all the radioactive substrates was a prominent feature in the head homogenate, being twice as great as the corresponding incorporations of radiocarbon in the leg muscle and fat-body. All three tissues incorporated radioactivity from the labelled acetate and succinate into alanine, aspartate, and glutamate. Measurable amounts of labelled proline were also found in the tissue homogenates incubated with the labelled acetate, and similar results with this radioactive substrate in locust fat-body have been reported by CLEMENTS(1959). It is well known that all these amino acids can become labelled from acetate or succinate vi0 either pyruvate or the tricarboxylic acid cycle, and the present results support the general conclusion of KILBY and NEWLLE (1957) that amino-acid metabolism in the locust resembles that in higher animals. The relatively large incorporations of radiocarbon into glutamate and alanine in all three tissue homogenates suggest that transaminase enzymes involving these amino acids are particularly active in locust tissues. KILBY and NWILLE (1957) reported that locust fat-body catalysed transamination reactions between a-ketoglutarate and numerous amino acids, the alanine-glutamate and aspartate-glutamate transaminase activities being particularly prominent. BELLAMY (1958) found glutamine to be present in fat-body, integument, thoracic muscle, and head of the locust, but CLEMENTS (1959) did not detect the formation of radioactive glutamine in fat-body incubated with labelled acetate. In the present work the leg muscle, fat-body, and head homogenates incorporated isotope from the labelled acetate and succinate into glutamine, showing that glutamine synthetase activity was present in all three tissues. REES (1954) observed that a number of tricarboxylic cycle acid intermediates were oxidized by muscle homogenates and sarcosomes of L. m&atoria and suggested that a major part of the endogenous respiration in this species was mediated via the cycle. In the present work the incorporation of Cl4 from the labelled substrates into fumarate, malate, aspartate, citrate, and glutamate is good evidence that the tricarboxylic acid cycle is operative in the leg muscle, fat-body, and head tissues of S. gregaria. The integrity of this cyclic enzyme system in locust fatbody has been questioned because HEARFIELDand KILBY (1958) failed to demonstrate the presence of condensing enzyme, a-ketoglutarate oxidase, and succinic
W. J. W. HI-
468
ANDM. J. H. SMITH
dehydrogenase in this tissue. However, CLEMENTS(1959) found that fluoracetate inhibited fat-body respiration, showing that aconitase activity was present; this result also provided indirect evidence of the presence of the condensing enzyme system. This worker also showed that locust fat-body produced CYOs from labelled succinate but stated that the succinate oxidase system appeared to be labile on homogenization, as was also suggested by BELLAMY (19.58). The utilization of the labelled succinate by all three tissue homogenates in the present work showed that fat-body resembled leg muscle and head in containing enough succinate dehydrogenase activity to utilize over 70 per cent of the labelled succinate supplied (Table 3). However, fat-body differed from the other tissues in forming a large proportion of labelled malate, suggesting that the further metabolism of malate was proceeding less efficiently. Thus the apparent lability of the succinate oxidase system of locust fat-body during homogenization may be a reflection of a diminished metabolism of malate rather than a loss of the ability to metabolize succinate itself. It appears that malic dehydrogenase activity and not succinic dehydrogenase activity is the more labile when locust fat-body is homogenized. Achowle&ments-We are grateful to the Anti-Locust Research Centrc for providing the 1OCUSt8 and to the Medical Research Council for a grant toward8 the coet of the work. REFERENCES D. (1958) The structure and metabolic propertie8 of tissue preparation8 from Scidrtocercu gregati (desert locust). Biochmr. J. 70, 580-589. CANDY D. J. and &LBY B. A. (19S9) Site and mode of trehalose biosynthesis in the locust. Nature, Lond. 183,1594-1959. CLENIBNTSA. N. (1959) Studies on the metabolism of locust fat body. J. esp. Biol. 36, 665-675. GOMORIG. (1955) Preparation of buffer8 for use in enzyme studies. In Methods in EnzymoZogy (Ed. by COLOWICK S. P. and KAPLAN N. O.), 1, 143. Academic Press, New York; HEARFIELD D. A. H. and KILBY B. A. (1958) Enzymes of the tricarboxylic acid cycle and cytochrome oxidase in the fat body of the desert locust. Nature, Lond. 181, 546-547. KILBY B. A. and NEVILLE E. (1957) Amino-acid metabolism in locust tissues. J. exp. Biol. 34, 276-289. RHBS K. R. (1954) Aerobic metabolism of the muscle of Locusta migratoriu. Biochem. J. BELLAMY
58, 196-202. SMITH M. J. H. and MOST V. (1960) Uncoupling reagent8 and metabolism.-I. The effects of salicylate and 2:4_dinitrophenol on the incorporation of l*C from labelled glucose and acetate into the soluble intermediates of isolated rat tissues. B&hem. J. 76, 579-585. WINTERINGHAMF. P. W. and LEWIS S. E. (1959) On the mode of action of insecticides.
Annu. Rev. Ent. 4. 303-318.
Printed in Great Britain
SOME ASPECTS OF INTERMEDIARY METABOLISM IN THE DESERT LOCUST (SCHISTOCERCA GREGARIA FORSIdL) W. J. W. HINES
and M. J. H. SMITH
Department of Chemical Pathology, Ring’s College Hospital Medical School, Denmark Hill, London (Received 2 March 1963)
Abstract-The incorporation and distribution of radioactivity from [“Cl glucose, [2-i%] acetate, and [l :4-“CI] succinate into the soluble metabolic intermediates of homogenates of the fat-body, head, end leg muscle of Schistocerca gregaria ForskHlhave been studied. The tissue homogenates were found to utilize the labelled substrates, but the patterns of incorporation of the radioactivity from each substrate varied with the tissue. INTRODUCTION
THE development of many insecticides during the last decade has stimulated interest in their modes of action. WINTERINGHAMand Lrrwrs (1959) have rightly emphasized that the exact manner by which an insecticide causes the death of a particular insect may be due to multiple biochemical and physiological actions rather than to some unique single lesion. It is therefore desirable to investigate as many as possible of the effects of such compounds on insect metabolism and to attempt to define them in biochemical terms. A necessary prerequisite is a detailed knowledge of insect biochemistry, but the study of this field has tended to lag behind corresponding work with mammalian tissues and micro-organisms. In recent years, the development of chromatographic methods for the separation of metabolic intermediates and the availability of radioactively labelled substrates have greatly facilitated the study of intermediary metabolism. This paper is concerned with the incorporation and subsequent distribution of radiocarbon from labelled glucose, acetate, and succinate amongst the soluble metabolic intermediates of the fat body, the nervous tissue of the head, and the leg muscle of the desert locust, Schisiocerca gregaria Forskil. MATERIALS AND METHODS The desert locust was chosen because of its large size and also because many individuals could be obtained on a single occasion from a standard population. Immature male and female insects were obtained from the Anti-Locust Research Centre, London, about 10 days after the final ecdysis and used at once. All dissections were carried out on cracked ice, and subsequent operations were performed as close to 0°C as possible. The heads were removed with scissors, the
W. J. W. Hmzs ANDM. J. H. SMITH
464
mouth-parts were cut off, and the remainder of the head was chopped up finely and homogenized with 4 volumes of cold 0.25 M sucrose in a Waring blendor. The femur muscle was removed from the hind legs as completely as possible, cut finely with scissors, and homogenized as above. The abdomen was cut open longitudinally along the mid-dorsal line, and the perivisceral fat body removed from either side of the gut with a pair of forceps and homogenized in 2 volumes of cold 0.25 M sucrose as above. Samples (50 ~1) of the homogenates were added to 75 ~1 of a solution containing 1 PC of the radioactive substrate, 5 mM MgCl,. m 6H,O, 0.15 mM adenosine triphosphate, dissolved in O-1 M phosphate buffer, pH 7.5 (GOMORI, 1955). The [‘“Cl glucose, [2-14C] acetate, and [l YI-‘~C~ succinate were obtained from the Radiochemical Centre, Amersham, Bucks. The mixtures were incubated aerobically in stoppered 5 ml tubes at 30°C with shaking for 60 min and the enzymes inactivated by the addition of 400 ~1 of boiling ethanol. After centrifuging for 5 min at 500g to remove denatured protein material, the radioactive substances in the supematants were separated by two-dimensional chromatography, visualized by radioautography, and the 14C was measured by the techniques described by SMITH and Moses (1960). RESULTS Metabolisnr of [‘“Cl glucose by locust tiwues The homogenates of all three tissues were capable of glucose (Table l), the leg muscle being the most active and as assessed by the amounts of residual radioactive glucose. corporated into trehalose by the three tissues ; they also
utilizing the labelled head the least active, Radiocarbon was informed a radioactive
TABLII I-DISTRIBUTION OF RADIOACTIVITY FROM [**Cl GLUCOSE IN SOLUBLE INTERMEDIATES OF HOMOGENATES OF LOCUSTTISSUES.&SULTs ARIIBXPRESSED AS COUNTSF%RMIN X 1o-3 Radioactive intermediate Trehalose Oligosaccharide Hexose phosphate Lactate Alanine Aspartate Glutamate Unidentified Glucose (residual substrate) Total in soluble fraction
Leg muscle
Tissue homogenate Fat-body
Head
0.1 3.5 4.8 1.0 5.6 0.1 0 0.1 5.8 15.2
1.1 1.2 2.9 0.4 3.4 4.1 0.1 0.5 9.8 13.7
1.0 3.9 0.6 0.4 3.4 0.3 1.3 0.3 13.9 11.2
oligosaccharide fraction which was not glycogen but produced on hydrolysis radioactive glucose only. The muscle and fat-body homogenates formed relatively large amounts of a hexose phosphate fraction, and ail the tissues incorporated
SOME ASPECTS OF INTERMEDIARY
METABOLISM
IN THE DESERT LOCUST
465
the isotope into lactate and alanine. The ratios alanine : lactate were approximately 6:l for the muscle and 10: 1 for the other tissues. Radioactivity was also detected in aspartate, and the fat-body and the head homogenates also formed radioactive glutamate. Metabolism of [2-‘4C]
acetate by locust tissues
The distribution of radioactivity from the labelled acetate amongst the soluble intermediates of the locust tissue homogenates is given in Table 2. The general pattern of incorporation of the isotope was similar for all three tissues except that the head homogenate formed a much greater proportion of labelled glutamate. TABLE SOLUBLE
2--DISTRIBUTION METABOLIC &SIJLTS
OF
RADIOACTIVITY
INTBRMEDIATBS
[2-“c]
FROM
OF HOMOGENATES
ARE BXPRBSSED AS COUNTS PER MIN
Radioactive intermediate Citrate Succinate Furnarate Malate Aspartate Glutamate Glutamine Alanine Lactate Proline Unidentified Total in soluble fraction
ACETATE
OF LOCUST
IN
TISSUES.
X 1o-3
Leg muscle
Tissue homogenate Fat-body
Head
O-1 0.2 0.1 0.8 0.1 4.3 o-2 l-2 l-5 0.5 0.1 9.1
O-1 0.5 0.3 1.8 0.1 2.0 0.1 0.2 1.2 0.1 0.1 6.5
0.4 1.0 0.3 1.5 0.1 9.5 0.8 1.1 3.5 0.2 0.2 18.6
Radioactivity was found in lactate and alanine, in acids of the tricarboxylic acid cycle (citrate, succinate, fumarate, and malate), and in amino acids (glutamate and aaparate), which were probably formed by transamr ‘nation reactions from their corresponding a-keto acids (a-ketoglutarate and oxaloacetate) which are also components of the tricarboxylic acid cycle. Labelled glutamine and proline were also detected in all the tissue homogenates. Metabolism of [ 1: 4-14CsJ succinate by locust times The results with the labelled succinate (Table 3) show that all three tissue homogenates were capable of utilizing this substrate by succinic dehydrogenase and metabolizing it via the tricarboxylic acid cycle. Radioactivity was found in fumarate, malate, citrate, aspartate, and glutamate. The presence of labelled glutamine confirmed the findings with the labelled acetate that glutamine synthetase activity occurred in all three tissues. The incorporation of radiocarbon into alanine and a phosphate fraction suggested that the succinate carbons had been converted to pyruvate presumably via the decarboxylation of malate or 3*
W. J. W. HINES AND M. J. H. SMITH
466 oxaloacetate.
The head homogenate again formed the largest amount of radio-
active glutamate, as in the acetate experiments, and the fat-body showed a relatively large incorporation of Cl4 into malate. TABLE %---DISTIXIBUTION OF RADIOACTIVITV FROM[t :4-“&J SUCCINATE IN IATESOF HOMOGENATES OF LOCUSTTISSUES. SOLUBLEMSTABOLICINTBRMBD &ISULTSAREEXPRESSED AS COUNTS PER MIN X lo-* Radioactive intermediate
Leg muscle
Tissue homogenate Fat-body
Head
Fumarate Malate Citrate Aspartate Glutamate Glutamine Alanine Phosphates Unidentified Succinate (residual substrate) Total in soluble fraction
1.4 4.8 0.2 0.4 2.8 0.4 2.7 0.1 0.1 20.5 33.4
3.4 16.2 0.1 0.3 2.3 0.1 2.2 0.1 0.1 25.5 50.3
0.8 4.4 0.5 0.3 5.9 0.7 2.1 0.1 0.2 15.6 30.6
DISCUSSION Studies
of isotope incorporation can provide useful data about the biochemical
pathways and the metabolic capabilities and relative importance of different tissues. Thus
the present results show that homogenates
metabolize
of locust tissues are able to
a wide range of radioactive substrates by established pathways
of
intermediary metabolism.
However, the patterns of incorporation of the radiocarbon amongst the soluble intermediates show differences between the tissues. Previous work in the locust using labelled glucose as a substrate has been almost entirely restricted to preparations of fat-body.
CLEMENTS (1959) found that
sheets of S. gregaria fat-body incorporated radiocarbon from r4C] glucose mainly into trehalose, smaller amounts being incorporated into glycogen, fat, and into an unidentified substance which was not glucose, ribose, alanine, or glutamate. CANDY and KILBY (1959) reported similar results and showed that the in Z&W biosynthesis of trehalose from glucose was increased by the addition of adenosine triphosphate
and uridine diphosphate
glucose.
They
also stated that a number
of unidentified radioactive compounds were formed from labelled glucose by locust fat-body tissue. The present results (Table 1) show that leg muscle and head homogenates are also capable of forming trehalose from glucose. In addition, radioactivity was incorporated into an oligosaccharide fraction, and all three tissue homogenates formed labelled hexose phosphates, lactate, and alanine. These findings indicate that as well as converting the glucose into the disaccharide, the locust tissues are capable of glycolytic reactions, and the pyruvate formed may be transaminated to alanine and acted on by lactate dehydrogenase to yield lactate.
SOME ASPECTS OF INTJ3RMEDIAFtY METABOLISM
IN THE DESERT LOCUST
467
The transamination reaction appeared to be quantitatively more important because the ratios of radioactive alanine to lactate varied from approximately 6:l to 10: 1, depending on the tissue. The muscle homogenate incorporated higher proportions of the radioactivity into the labelled intermediates (hexose phosphates, alanine, and lactate), associated with glycolysis, than did the other tissues. Radioactive aspartate was found in all the tissue homogenates, particularly the fat-body, and the carbon skeleton of this amino acid could be derived either via the carboxylation of pyruvate to oxaloacetate and subsequent transamination or after the entry of pyruvate carbons into the tricarboxylic acid cycle. The presence of labelled glutamate in the fat-body and head homogenates provides good evidence that glucose carbons had entered the tricarboxylic acid cycle, presumably via pyruvate and acetylcoenzyme A, to form a-ketoglutarate which was transaminated to glutamate. The formation of labelled glutamate from all the radioactive substrates was a prominent feature in the head homogenate, being twice as great as the corresponding incorporations of radiocarbon in the leg muscle and fat-body. All three tissues incorporated radioactivity from the labelled acetate and succinate into alanine, aspartate, and glutamate. Measurable amounts of labelled proline were also found in the tissue homogenates incubated with the labelled acetate, and similar results with this radioactive substrate in locust fat-body have been reported by CLEMENTS(1959). It is well known that all these amino acids can become labelled from acetate or succinate vi0 either pyruvate or the tricarboxylic acid cycle, and the present results support the general conclusion of KILBY and NEWLLE (1957) that amino-acid metabolism in the locust resembles that in higher animals. The relatively large incorporations of radiocarbon into glutamate and alanine in all three tissue homogenates suggest that transaminase enzymes involving these amino acids are particularly active in locust tissues. KILBY and NWILLE (1957) reported that locust fat-body catalysed transamination reactions between a-ketoglutarate and numerous amino acids, the alanine-glutamate and aspartate-glutamate transaminase activities being particularly prominent. BELLAMY (1958) found glutamine to be present in fat-body, integument, thoracic muscle, and head of the locust, but CLEMENTS (1959) did not detect the formation of radioactive glutamine in fat-body incubated with labelled acetate. In the present work the leg muscle, fat-body, and head homogenates incorporated isotope from the labelled acetate and succinate into glutamine, showing that glutamine synthetase activity was present in all three tissues. REES (1954) observed that a number of tricarboxylic cycle acid intermediates were oxidized by muscle homogenates and sarcosomes of L. m&atoria and suggested that a major part of the endogenous respiration in this species was mediated via the cycle. In the present work the incorporation of Cl4 from the labelled substrates into fumarate, malate, aspartate, citrate, and glutamate is good evidence that the tricarboxylic acid cycle is operative in the leg muscle, fat-body, and head tissues of S. gregaria. The integrity of this cyclic enzyme system in locust fatbody has been questioned because HEARFIELDand KILBY (1958) failed to demonstrate the presence of condensing enzyme, a-ketoglutarate oxidase, and succinic
W. J. W. HI-
468
ANDM. J. H. SMITH
dehydrogenase in this tissue. However, CLEMENTS(1959) found that fluoracetate inhibited fat-body respiration, showing that aconitase activity was present; this result also provided indirect evidence of the presence of the condensing enzyme system. This worker also showed that locust fat-body produced CYOs from labelled succinate but stated that the succinate oxidase system appeared to be labile on homogenization, as was also suggested by BELLAMY (19.58). The utilization of the labelled succinate by all three tissue homogenates in the present work showed that fat-body resembled leg muscle and head in containing enough succinate dehydrogenase activity to utilize over 70 per cent of the labelled succinate supplied (Table 3). However, fat-body differed from the other tissues in forming a large proportion of labelled malate, suggesting that the further metabolism of malate was proceeding less efficiently. Thus the apparent lability of the succinate oxidase system of locust fat-body during homogenization may be a reflection of a diminished metabolism of malate rather than a loss of the ability to metabolize succinate itself. It appears that malic dehydrogenase activity and not succinic dehydrogenase activity is the more labile when locust fat-body is homogenized. Achowle&ments-We are grateful to the Anti-Locust Research Centrc for providing the 1OCUSt8 and to the Medical Research Council for a grant toward8 the coet of the work. REFERENCES D. (1958) The structure and metabolic propertie8 of tissue preparation8 from Scidrtocercu gregati (desert locust). Biochmr. J. 70, 580-589. CANDY D. J. and &LBY B. A. (19S9) Site and mode of trehalose biosynthesis in the locust. Nature, Lond. 183,1594-1959. CLENIBNTSA. N. (1959) Studies on the metabolism of locust fat body. J. esp. Biol. 36, 665-675. GOMORIG. (1955) Preparation of buffer8 for use in enzyme studies. In Methods in EnzymoZogy (Ed. by COLOWICK S. P. and KAPLAN N. O.), 1, 143. Academic Press, New York; HEARFIELD D. A. H. and KILBY B. A. (1958) Enzymes of the tricarboxylic acid cycle and cytochrome oxidase in the fat body of the desert locust. Nature, Lond. 181, 546-547. KILBY B. A. and NEVILLE E. (1957) Amino-acid metabolism in locust tissues. J. exp. Biol. 34, 276-289. RHBS K. R. (1954) Aerobic metabolism of the muscle of Locusta migratoriu. Biochem. J. BELLAMY
58, 196-202. SMITH M. J. H. and MOST V. (1960) Uncoupling reagent8 and metabolism.-I. The effects of salicylate and 2:4_dinitrophenol on the incorporation of l*C from labelled glucose and acetate into the soluble intermediates of isolated rat tissues. B&hem. J. 76, 579-585. WINTERINGHAMF. P. W. and LEWIS S. E. (1959) On the mode of action of insecticides.
Annu. Rev. Ent. 4. 303-318.